The objective of work proposed in this application is to decipher important new mechanisms that trigger the development of human cancer. Among human adenoviruses, adenovirus type 9 is unique in causing exclusively estrogen-dependent mammary tumors in experimental animals and in having E4 region-encoded open reading frame 1 (E4-ORF1), rather than E1 region-encoded E1A and E1B, as its primary oncogenic determinant. Evidence indicates that the tumorigenic potential of E4-ORF1 depends on two separate protein-interaction elements, the PDZ domain-binding motif (PBM) and Domain 2 (D2). While D2 mediates binding to several unidentified cellular proteins, the PBM interacts with a select group of cellular PDZ proteins, including the three cell polarity proteins Dlg1, ZO-2, and PATJ, as well as the PATJ paralogue MUPP1 and MAGI-1. Underscoring the link of these cellular PDZ proteins to human cancer, Dlg1, PATJ, MUPP1, and MAGI-1 are also cellular targets of high-risk human papillomavirus E6 oncoproteins and Dlg1 is a cellular target of the human T-cell leukemia virus type 1 Tax oncoprotein. Our results lead to the hypothesis that the tumorigenic potential of E4-ORF1 stems from its combined capacities to induce Dlg1- and D2 protein-dependent constitutive PI3K activation and to block both PATJ- and ZO-2-dependent apical-basal cell polarity establishment and Dlg1-dependent anterior-posterior cell polarity establishment. This hypothesis will be tested by (Aim 1) purifying, identifying, and characterizing D2-interacting proteins and determining their role in disregulated PI3K signaling, (Aim 2) investigating tumor suppressor functions for PATJ and ZO-2 in mice, and (Aim 3) determining mechanisms for E4-ORF1-induced inhibition of anterior-posterior cell polarity. The immediate benefits of completing these aims will be the elucidation of novel mechanisms leading to tumorigenic activation of the PI3K pathway and loss of cell polarity. As both defects are hallmarks of cancer cells, this information should provide important new insights into the development of a wide variety of human malignancies. In the long-term, the knowledge gained from the proposed work may aid in the development of effective new therapeutic strategies to treat and prevent human cancers.